Isolation, identification and phylogenetic analysis of a pathogen of Haliotis diversicolor supertexta (L.) with mass mortalities Item Type article Authors Zhou, Jing; Cai, Junpeng Download date 29/09/2021 14:57:43 Link to Item http://hdl.handle.net/1834/37408 Iranian Journal of fisheries Sciences 12(4)843-854 2013 Isolation, identification and phylogenetic analysis of a pathogen of Haliotis diversicolor supertexta (L.) with mass mortalities Zhou Jing*1and Cai Junpeng 2 Received: February2013 Accepted: Abstract This study was conducted to determine a disease outbreak in 14 day old post-larvae of abalone ( Haliotis diversicolor supertexta ) which caused mass mortality in July 2010 in Shanwei, China. Twenty-nine bacterial strains were isolated from a sample pool of 10 diseased post-larval abalones on 2216E marine agar plates during a natural outbreak of the disease. Among them, a dominant isolate (referred to as strain 21) was found to be highly 4 virulent to post-larvae in experimental challenge tests, with an LD 50 value of 1.0 ×10 colony forming units (CFU) mL -1 on day 3. API 20NE kits and 16S rDNA sequence analysis, identified strain 21 as Oceanomonas doudoroffii. It was susceptible to 10 and Downloaded from jifro.ir at 16:31 +0330 on Saturday February 17th 2018 moderately susceptible to 1 of the 16 antibiotics examined when antibiotic sensitivities of the bacterium were assayed. Results of this study implicated Oceanomonas doudoroffii strain 21 as a cause of mortalities in post-larval abalone from Shanwei, China. Keywords : Haliotis diversicolor supertexta , massive death, challenge test, Oceanomonas doudoroffii , 16S rDNA sequencing 1-College of Life Science, QuFu Normal University, QuFu, China 2-College of Food Science and Light Industry, South China University of Technology, Guangzhou, China Corresponding author’s email: [email protected] Iranian Journal of fisheries Sciences 12(4) 2013 844 Introduction Production of various species of the genus when the post-larvae were just 14 days post Haliotis is a very important part of aquaculture in fertilization. Post-larvae were on average the Orient since they have been regarded as very approximately 0.3mm in length. To maximize precious seafood. However, from 2002 onwards, chances of bacterial isolation, ten specimens mass mortalities of post-larvae of the small were collected with a pipette fitted with a sterile abalone, Haliotis diversicolor supertexta , have 1-mL tip from bio-films, and placed in a sterile persisted and forced many abalone farms to be polystyrene Petri dish containing 0.5 mL sterile closed (Cai et al., 2006a). Diseased post-larval phosphate buffered saline (PBS) of 0.01 abalone became pale and lethargic with atrophied mol/L(pH 7.2). Then they were transferred to muscles. Most of the post-larvae came off the the Microbiological Laboratory and diatom bio-films on which they grew one to two homogenized in a sterile glass grinder with 0.5 days after these symptoms appeared. Some did mL sterile PBS after being rinsed 3 times with not come off the bio-films but their shells were PBS. A 10-fold dilution series ranging from 10 -1 left empty. to 10 -5 was prepared from this homogenate. Upon examination of the cause(s) of these Each sample in the dilution series was plated outbreaks, Cai et al. (2006a,b,c) revealed that out on 2216E Marine (Difco, supplemented Vibrio parahaemolyticus, Shewanella algae , V. with 2.5% NaCl) agar plates using 0.1 mL alginolyticus and Klebsiella oxytoca were all inoculum per plate. After 4-6 days incubation at associated with mass mortality of post-larval 25 °C, representative colonies, according to abalone in Shanwei and Fujian. However, it is their different morphologies and abundance on not yet known if any of these pathogens were also the culture media, were selected and purified for Downloaded from jifro.ir at 16:31 +0330 on Saturday February 17th 2018 responsible for other outbreaks in different farms characterization and identification. in the same region. We report the isolation and Abalone and virulence test characterization of Oceanomonas doudoroffii as a Abalone of 20 days post-fertilization with an cause of mass mortalities in the post-larval average shell length of ca. 0.9 mm were collected abalone, H. diversicolor supertexta in July 2010 from an abalone farm in Shenzhen, Guangdong in Shanwei, China. Province. There was no incidence of disease outbreaks at this site during the hatching season. Materials and methods These abalone were used for experimental Bacterial isolation challenge tests which were run according to the The whitened (diseased) post-larval abalone, protocol described by Cai et al. (2006a). Briefly, that used for the bacterial isolation were from an each post-larval abalone, with its attached diatom abalone farm in Shanwei of Guangdong film, was first cut into pieces of approximately 1 Province, China. They were collected during an cm 2 or less and placed in a sterile 2-L beaker and outbreak of post-larval disease in July 2010 rinsed 3 times with 500 mL autoclaved and 845 Jing and Junpeng ., Isolation, identification and phylogenetic analysis of a pathogen … aerated seawater containing six antibiotics if it was recovered in pure culture from dead (Takara, China)(chloramphenicol, 20 mg/ L; post-larvae (Brock et al., 1994). norfloxacin, 10 mg/L; erythromycin, 15 mg/L; Bacterial characterization gentamicin, 40 mg/L; penicillin G, 200,000 IU/L; The pure cultures recovered from the sample and polymyxin B, 300 mg /L) and then immersed pool of 10 diseased post-larval abalone were in 1 L of the same mixed antibiotic solution for a checked to establish if they could grow on day with change of the water every 6 h. Once thiosulfate citrate bile salt sucrose medium (TCBS; pour plate technique had confirmed bacteria-free Difco, Detroid, USA) supplemented with 2.5 % status, challenge tests were carried out. A series of (w/v) NaCl. Further experiments were carried out dilutions of 4 different bacterial suspensions (24 h only to characterize the most virulent bacterial bacterial culture, 10 3–10 6 CFU/mL, final strains identified in the challenge tests. concentrations) was run in the tests with 2-L The most abundant and virulent bacterial strain beakers as containers (Trevors and Lusty, 1985). (viz. strain 21) was subjected to standard To each 2-L beaker, 1L of autoclaved, aerated 3% morphological, physiological, and biochemical salinity sand-filtered seawater, 20 bacteria-free examination. Gram-reaction, oxidase, catalase and post-larval abalones and the appropriate hydrolysis of aesculin and gelatin were tested as concentration of bacteria were added. Three described by Baumann et al. (1972) and Smibert beakers without any added bacteria were used as and Krieg (1994). The ability to use phenol as the negative controls. sole carbon source was determined on minimal All twenty-nine isolates, recovered from the media that contained 2% NaCl (w/v) and 4 mM diseased abalone homogenate, were subjected to phenol for up to 7 days incubation. Leifson’s challenge tests, performed over a three-day period oxidation–fermentation medium for marine Downloaded from jifro.ir at 16:31 +0330 on Saturday February 17th 2018 and in triplicate. LD 50 values were calculated on bacteria (Leifson, 1963) was used to test acid 8 -1 day 3. LD 50 values of >10 CFU mL were production from carbohydrates with 1% (w/v) of considered a-virulent, and values of between 10 4 each compound. Other biochemical tests were and 10 5 CFU/mL were considered virulent, while carried out using API 20NE test kits (ATB system, values between 10 6 and 10 7 CFU mL -1 were BIOMÉRIEUX SA, Marcy-I’Etoile, France) considered weakly virulent in line with the according to the manufacturer’s instructions. virulence criteria of Mittal et al. (1980). In order to characterize strain 21 at the To verify Koch’s postulate in the case of the molecular level, 16S and its rDNA sequencing most virulent strain, each of the post-larvae in the and phylogenetic analyses was performed. Strain challenge tests was picked-up under a magnifier, 21 was grown overnight at 25 °C in marine broth thoroughly rinsed with sterile PBS and used for with shaking at 180 rpm. Cells from cultures were re-isolation and identification of bacteria. harvested by centrifugation at 10,000 g for 2 min, Mortality was attributed to the bacterium isolated rinsed, and then suspended in 1x TE buffer (10 mM Tris-HCl, 100 mM EDTA, pH 8.0). Genomic Iranian Journal of fisheries Sciences 12(4) 2013 846 DNA extraction and PCR amplification of 16S Escherichia coli ATCC 25922 was also rDNA of strain 21 were run as reported by Cai et included in the analysis as a control bacterium. al. (2006b). After confirmation of successful Antibiotic sensitivity to a particular antibiotic (i.e. amplifications by electrophoresis of 5 µL PCR sensitive, intermediately sensitive or resistant) products on a 1% agarose gel, the products were was assessed according to the recommended purified using a PCR purification kit (Takara, cut-off levels of the zone size. China). PCR direct sequencing was done as Results reported by Thompson et al. (1992). Sequence Twenty-nine representative colonies were data was then deposited in GenBank. The isolated from sampled diseased post-larval computer program Blast was used to identify the abalone during the disease outbreak in July 2010 species which was phylogenetically closest to the in Shanwei. Selection of these colonies was based sequence of strain 21 and an evolutionary tree was on differences in morphology and their relative determined using the Neighbor-Joining method abundances on marine agar plates. One isolate, (Saitou and Nei, 1987) with the program EGA designated as strain 21, was found to be the version 4.0.